We performed hybridizations between labeled rRNAs from seven representative members of the family Pasteurellaceae and from three other taxa on the one hand and DNAs from 53 strains known or presumed to belong to the Pasteurellaceae on the other hand. The members of the Pasteurellaceae are most closely related to members of the Enterobacteriaceae, the Vibrionaceae, the Aeromonadaceae, and the genus Alteromonas. The family Pasteurellaceae is very heterogeneous. There are at least seven rRNA branches. Several organisms with the same genus name are dispersed over the entire dendrogram. The "Histophilus ovis," [Haemophilus] ducreyi, [Actinobacillus] actinomycetemcomitans, and [Haemophilus] aphrophilus rRNA branches are separate and quite remote from the three authentic genera in this family; this might justify eventual later separate generic status. DNA-rRNA hybridization with suitable, labeled rRNA probes is an excellent method to establish whether an organism belongs in the Pasteurellaceae; e.g., some strains of Bisgaard's taxa 7, 13, and 16 and of the gas-producing "SP" group certainly belong in this family, whereas three bovine lymphangitis organisms (strains NCTC 10547, NCTC 10549, and NCTC 10553), [Haemophilus] piscium ATCC 10801T (T = type strain), and [Pasteurella] piscicida ATCC 17911 belong in the Enterobacteriaceae, the Aeromonadaceae, and the Vibrionaceae, respectively.

The aim of this study was to reinvestigate the relationships and the generic affiliations of the species Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus, Haemophilus paraphrophilus and Haemophilus segnis. The nicotinamide phosphoribosyltransferase gene (nadV) conferring V factor-independent growth was identified in Haemophilus aphrophilus. The gene encodes a polypeptide of 462 amino acids that shows 74.5 % amino acid sequence identity to the corresponding enzyme from Actinobacillus actinomycetemcomitans. Ten isolates of Haemophilus paraphrophilus all carried a nadV pseudogene. DNA from Haemophilus aphrophilus was able to transform Haemophilus paraphrophilus into the NAD-independent phenotype. The transformants carried a full-length nadV inserted in the former locus of the pseudogene. The DNA-DNA relatedness between the type strains of Haemophilus aphrophilus and Haemophilus paraphrophilus was 77 %. We conclude that the division into two species Haemophilus aphrophilus and Haemophilus paraphrophilus is not justified and that Haemophilus paraphrophilus should be considered a later heterotypic synonym of Haemophilus aphrophilus. Forty strains of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus segnis were investigated by multilocus sequence analysis. The 40 strains form a monophyletic group clearly separate from other evolutionary lineages of the family Pasteurellaceae. We propose the transfer of Actinobacillus actinomycetemcomitans, Haemophilus aphrophilus and Haemophilus segnis to a new genus Aggregatibacter gen. nov. as Aggregatibacter actinomycetemcomitans comb. nov. (the type species; type strain ATCC 33384(T)=CCUG 13227(T)=CIP 52.106(T)=DSM 8324(T)=NCTC 9710(T)), Aggregatibacter aphrophilus comb. nov. (type strain ATCC 33389(T)=CCUG 3715(T)=CIP 70.73(T)=NCTC 5906(T)) and Aggregatibacter segnis comb. nov. (type strain HK316(T)=ATCC 33393(T)=CCUG 10787(T)=CCUG 12838(T)=CIP 103292(T)=NCTC 10977(T)). The species of the genus Aggregatibacter are independent of X factor and variably dependent on V factor for growth in vitro.